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ROLE OF GLU96, ASP1O2 AND ASP111 IN FACTOR V
Coagulation factor V activity is unmasked by thrombin-mediated excision
of the central B domain resulting in a noncovalent heterodimer, factor Va. To
understand the role of individual amino acids in maintaining the Ca²⁺-depenadent
subunit interaction, G1u96 (E96A), Asp1O2 (D1O2A) or Asp111 (D111A) were
mutated because of known effects on chelator sensitivity. The primary clotting
activity of each mutant was reduced by “40%. Demonstrating at least two
distinct inhibition mechanisms, only D111A was further inhibited by thrombin
pre-treatment consistent with spontaneous subunit dissociation and severely
inhibited Ca²⁺ binding. The parental factor V construct used here has a truncated
B domain that does not require excision for activity. Therefore inhibition of
D111A by thrombin-cleavage reveals a new B domain function that maintains
factor V in a factor Va-like configuration independent of Ca²⁺ binding. In addition
to Ca²⁺, factor V binds Cu²⁺, but with unknown function. Unexpectedly, D111A
also lost detectable Cu²⁺. Finding that a single amino acid substitution
simultaneously alters Ca²⁺ and Cu²⁺ suggests an interdependent metal ion
binding site. Unlike D111A, the thrombin-mediated factor Va derived from E96A
and D1O2A was stable, had only moderately faster subunit dissociation upon
chelation and had normal metal ion binding. Thus, the current study defines the
highly conserved acidic segment spanning G1u96-Asp112 in factor V as
multifunctional. Of the three amino acids I evaluated, Asp111 is essential and
likely functions through direct and indirect metal ion interactions. G1u96 and
Asp102 individually influence factor V/Va function by more subtle effects at the
metal ion-dependent subunit interface.
FACTOR V-DEFICIENT PATIENT
A factor V-deficient patient due to Y1702C mutation has been studied. The
patient however did not suffer from severe bleeding despite of undetectable
levels of plasma and platelet factor V. A close inspection of the patient’s blood
coagulation cascade showed that the lack of available factor V was compensated
by other factors that influence the intrinsic pathway. This finding suggests that
the commonly observed phenotypic differences shown among factor V-deficient
patients with the same genotypes may be due to existing hypercoagulant factors
that influence the outcome of the disease.

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